Finding of Cyclodextrans and Attempts of their Industrialization for Cariostatic Oligosaccharides

Abstract

Cyclodextrans (CIs) are cyclic oligosaccharides consisting of Dglucoses bound through α(16) linkages produced from dextran catalyzed by cyclic isomaltooligosac-cahride glucanotransferase (or cyclodextran glucanotrans-ferase, CITase). 1) In the beginning, CIs were expected to be a novel dominant candidate for making inclusion complexes with some larger components or highly hydropho-bic components that hardly ever make a complex with cy-clodextrins (CDs) because CIs are highly watersoluble and have larger cavities in the center than that of CDs. However, no significant inclusion ability comparable to that of CDs has been found in CIs. 2) On the other hands, CIs strongly inhibit glucansucrase activity, 3) which is thought useful in preventing dental caries, while CDs do not have this property. The cariostatic activity of CI is much stronger than that of palatinose 3) or polyphenol. 4) We have been doing research and development on CI production to utilize CI as a novel dental caries inhibitor. Isolation of cyclodextran(CI)-producing bacterial strains and structural determination of CI. Dextran digestive strains were isolated from soils, and in the culture supernatant of Bacillus circulans T3040 strain when it was cultivated with dextran, three unknown peaks were detected by HPLC analysis (Fig. 1). 1) Those oligosaccharide peaks a, b and c were collected and purified. They were digested by endodextranase but not by exodextranase, suggested they were α1,6 linked glucose residues but not linear in form. 13 C-NMR and mass analyses were done on the purified oligosaccharides a, b and c. As shown in Fig. 2, the chemical shifts of 13 C-NMR spectra of C1, C2, C3, C4, C5 and C6 of the isolated oli-gosaccharides indicated the same signals of glucopyla-noside (6Glcp) observed in α1,6 glucan (dextran). 13 C-NMR analysis also showed all the isolated oligosaccha-rides consisted of 6Glcp. Mass spectra analysis was also done. As shown in Fig. 3, [M+H] ions of 1135.38, 1297.43 and 1459.48 m z were observed in the isolated products. Molecular masses of isomaltoheptaose, isomaltooctaose and isomalto-nonaose are calculated as 1152, 1302 and 1476, respectively. However, molecular masses of the isolated sugars were calculated as 1134, 1296 and 1458, respectively, which are 18 (the molecular mass of one H2O) smaller than those of isomaltooligosaccharides consisting of the same molecular numbers of glucose units. That is to say, the novel sugar products produced by B. circulans T3040 strain were cyclic isomaltooligosaccharides (cyclodextrans, CI). We identified them as cycloisomaltoheptaose, cyclo-isomaltooctaose, and cycloisomaltononaose and named them CI7, CI8 and CI9, respectively. In addition, larger CIs at least up to CI17, were also observed in the CI producing bacterial strain culture supernatant when it was grown with dextran. Abstract: Cyclic isomaltooligosaccharides or cyclodextrans (CIs) are cyclic oligosaccharides of α-1,6 linked glucose residues. CIs are highly water-soluble and were found to strongly inhibit glucansucrase activity of mu-tans streptococci, so, CIs are expected to be utilized as cariostatic compounds. They are produced from dex-tran catalyzed by cyclic isomaltooligosaccharide glucanotransferase (CITase) and substrate dextran is produced from sucrose catalyzed by dextransucrase. CIs were found and isolated from the culture supernatant of Bacillus circulans T-3040 strain when it was cultured with dextran. The structure of CIs were determined by enzyme digestion test, 13 C-NMR analysis, and mass spectrum analysis. In order to produce CIs for commercial scale, the high dextran producing strain Leuconostoc sp. S-51 was isolated and the B. circulans T-3040 strain was mutated to produce about 110 times as much CITase as that of wild type strain. We also successfully detected CIs in brown sugar, which suggests CIs exist in nature.